Simple, fast and inexpensive quantification of glycolate in the urine of patients with primary hyperoxaluria type 1

In primary hyperoxaluria type 1 excessive endogenous production of oxalate and glycolate leads to increased urinary excretion of these metabolites. Although genetic testing is the most definitive and preferred diagnostic method, quantification of these metabolites is important for the diagnosis and evaluation of potential therapeutic interventions. Current metabolite quantification methods use laborious, technically highly complex and expensive liquid, gas or ion chromatography tandem mass spectrometry, which are available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with glyoxylate generated from glycolate using recombinant mouse glycolate oxidase (GO) and glycine leads to the formation of a stable dihydroquinazoline double aromatic ring chromophore with specific peak absorption at 440 nm. The urinary limit of detection and estimated limit of quantification derived from eight standard curves were 14.3 and 28.7 µmol glycolate per mmol creatinine, respectively. High concentrations of oxalate, lactate and L-glycerate do not interfere in this assay format. The correlation coefficient between the absorption and an ion chromatography tandem mass spectrometry method is 93% with a p value < 0.00001. The Bland–Altmann plot indicates acceptable agreement between the two methods. The glycolate quantification method using conversion of glycolate via recombinant mouse GO and fusion of oABA and glycine with glyoxylate is fast, simple, robust and inexpensive. Furthermore this method might be readily implemented into routine clinical diagnostic laboratories for glycolate measurements in primary hyperoxaluria type 1. Supplementary Information The online version contains supplementary material available at 10.1007/s00240-023-01426-6.

3 ultracentrifugation tubes with a molecular weight cut-off of 30 kDa and protein concentration was determined using the 448 nm extinction coefficient of GO of 9200 M -1 cm -1 [22]. Concentrated protein was flash-frozen as protein droplets in liquid nitrogen and stored at -80 °C until further use. In all presented GO assay data 1 µM (43 µg/mL) final GO concentration was used after thawing a protein droplet. At this concentration the GO absorption at 440 nm was 0.0120 and this value was subtracted from the samples containing GO. Thawed GO protein droplets were used only once within two hours after thawing. We also tested human and spinach GO in the same expression system, but mouse GO showed the highest expression levels and most stable protein expression and was selected for purification (data not shown).

Measurement of glycolate using mass spectrometry
The Dionex Integrion HPIC coupled with an ISQ EC single quadrupole mass spectrometer was equipped with a Dionex EGC III electrolytical KOH eluent generator cartridge with a Dionex CR-ATC 600 anion trap column, suppressor AERS 500e Integrion conductivity detector and a Dionex AS-AP autosampler. The Chromeleon chromatography data system software was used for analysis. Chromatographic separation was conducted at 30°C on Dionex IonPac AS11 (2×250 mm) and AG11 (2×50 mm) analytical and guard columns. All devices and software are from Thermo Fisher Scientific (Waltham, MA).
Urine collected at home over 24 hours was acidified with 6 mM HCl and transported to the laboratory within a few days. After arrival samples were analyzed within 24 hours in most cases. Urine samples (5 µl of a 1 to 100 dilution) were injected by the autosampler and a KOH multistep gradient was applied via the eluent generator cartridge with a flow rate of 0.3 mL/min. The gradient consisted of 5 to 40 µM (0-15 min), 40 to 100 µM (15-15.2 min), 100 µM (15.2-18 min), 100 to 5 µM (18-18.2 min) and 5 µM .
Electrospray ionization in the negative mode was used with the following settings: vaporizer 500 °C, ion transfer tube 200 °C, source voltage 2500 V, source collision-induced dissociation (CID) 20 V, sheath gas 50 psig, auxiliary gas 5 psig and sweep gas off. Glycolate was detected using selected ion monitoring (SIM) at 75 m/z and quantified using a glycolate standard curve. Standards and controls were prepared using the certified reference material Supelco glycolate standard for IC (1000 mg/L glycolate in water, TraceCERT, 07391, Sigma-Aldrich). Urine samples were diluted 1 to 100 in 0.3 M boric acid (B6768 BioReagent, ≥99.5%, Sigma-Aldrich).
The glycolic acid standard curve consisted of glycolic acid at 0, 0.2, 0.4, 1, 2 and 4 µM. Two quality control samples at 1.2 and 3 µM glycolate in 0.3 M boric acid were used. The method is specific and selective with the 4 calibration curve linear over the standard curve range tested using Mandel's F-test. Typical performance is reflected by a bias of -0.9 % with an intra-and inter-day precision of 9.8 % relative standard deviation (RSD) for both parameters. Samples of the ERNDIM External Quality Assurance Scheme for Special Assays in Urine are analyzed on a regular basis with satisfactory performance (https://www.erndim.org/).

Fragmentation MS2 analysis of the 235.071 m/z peak representing CCMDQ
Fragmentation of CCMDQ was performed using 20 V collision energy. The predicted and measured MS2 fragments are shown in Supplementary Fig. S1 and Table S1. The peak base in the stick diagram is 150 m/z (Fig.   S1B). Other prominent peaks are at 106 (numbered 1 in Supplementary Fig. S1), 118 (2), 128, 145 (3), 168 (5), 191 (7) m/z. Five of these peaks including the peak base (106, 118, 145, 150 (4) and 191 m/z) have been predicted (Supplementary Table S1).    After adding oABA, glycine and glyoxylate to urine samples, peak absorption is reached within 10 minutes ( Supplementary Fig. S4). After the peak the signal slowly degrades between 15 and 21 % in the four highest concentrations during the 60 minutes incubation time.  The area under the curve (AUC) was calculated from the samples shown in Fig. 3B; OX = sodium oxalate, LA = sodium lactate, GLY = sodium L-glycerate; GO = mouse glycolate oxidase without exogenous substrate; noGO = only buffer control added to urine; SD = standard deviation; Numbers after the letters in the first column represent metabolite concentrations in µM.

High concentrations of thymol inhibit signal generation in the GO assay
The urine samples in Cohort_2_PH should have been collected using a 1 in 100 dilution of Thymol 5% (2isopropyl-5-methylphenol in 100 % isopropanol; 12848.00100; Morphisto; Offenbach am Main, Germany).
Nevertheless, patients were advised to dilute Thymol 5% during collection of the 24 hour urine samples at home.
Therefore the final concentrations of thymol might vary considerably. Before we tested these urine samples in the GO assay we measured the influence of different thymol concentrations on the GO assay performance. If Thymol 5 % is properly diluted the final concentrations would be 0.05 % corresponding to 500 µg/mL or 3.3 mM thymol with 1% isopropanol. The GO inhibition data are summarized in Supplementary Fig. S5. Recombinant mouse GO starts to be inhibited between 0.05 % and 0.1 % thymol, but inhibition is not linear, with higher glycolate concentrations more inhibited compared to lower concentrations. Different Thymol 5 % dilutions might have been used for some patients´ urine samples, and therefore thymol must be measured in each urine sample to somewhat estimate GO inhibition. We used the Gibbs method to quantify thymol including other phenolics in the relevant urine samples (Gibbs 1927). Gibbs reagent 2,6-dichloroquinone-4-chloroimide condensates with phenol, phenolics (mainly p-and m-cresol) and also thymol generating a chromophore with absorption maximum at 610 nm. and 700 nm showed a thymol concentration dependent increase in absorption with a peak at 615 nm (data not shown).
Urine did not relevantly influence the Gibbs method compared to water ( Supplementary Fig. S7A) and a linear standard curve can be generated using 1 to 60 µg/mL thymol concentrations (Supplementary Fig. S7B and S7C).
The signal is stable for at least 90 minutes. Based on the data shown in Supplementary Fig. S7D Fig. S7D). A few thymol concentrations in PH samples were above 10 mmol/mmol creatinine ( Supplementary Fig. S7D).

Measurement of glycolate concentrations in Cohort_1_PH and Cohort_2_PH using the glycolate oxidase assay and IC-MS
Both cohorts are briefly described in Supplementary Table S5 and Table S6. The IC-MS method (glycolate µmol/mmol creatinine BON) used for these cohorts is described below.
Acidified samples can be measured with the GO method with a high correlation coefficient of 93 % and low pvalue of < 0.0001 ( Supplementary Fig. S8A). Samples containing mainly Thymol 5 % as preservative show a lower correlation coefficient of 73 % but still a significant p-value of 0.0082 ( Supplementary Fig. S8B).